The objectives of this study are to understand the nature of multiple allosteric interactions in the control of cellular processes, the role of subunit-subunit interactions, the manner in which these interactions are influenced by the binding of allosteric modifiers to enzyme systems and by amino acid substitutions in the protein. The test system is phosphoenolpyruvate carboxylase of Escherichia coli. This is an allosteric enzyme that plays a major role in the control of the flow of intermediary metabolites through the citric acid cycle. Its product, oxaloacetic acid, and its primary allosteric activator, acetyl-coenzyme A, are the substrates for the formation of citrate, the initial substrate of the cycle. Phosphoenolpyruvate carboxylase activity is also stimulated in vitro by nucleotide phosphates, by long chain fatty acids and their coenzyme A derivatives, and by fructose-1,6-diphosphate. The mechanisms of the interaction of these effectors on the enzyme singlely and in combination is studied primarily by kinetic methods. Partially active and inactive enzyme preparations purified from mutants of E. coli are used to identify which amino acid changes are responsible for the altered functions. The amino acid changes are determined by conventional methods on protein fragments obtained by specific enzymatic digestion or specific chemical cleavage. Attempts are made to form hybrids from active and inactive subunits of the enzyme to assess the relative importance of amino acid substitution in the enzyme's catalytic and regulatory functions, and to determine their role in subunit-subunit interaction.